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[Protective Effect of Acupuncture Serum Derived from Acute Convulsion Rats on Cultured Hip-pocampal Neurons with Seizure-like Discharges by Regulating Expression of Endoplasmic Reticulum Stress-inducible Molecular Chaperones].
Zhen Ci Yan Jiu. 2017 Apr 25; 42(2):95-101.ZC

Abstract

OBJECTIVE

To observe the effect of acupuncture serum on the number of apoptosis of the cultured hippocampal neurons with seizure-like discharges and the expression levels of endoplasmic reticulum stress-inducible molecular chaperones glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP) and cysteine protease protein-12(Caspase-12), so as to reveal its protective mechanism on seizure-induced injury of hippocampal neurons.

METHODS

A regular primary culture of neurons derived from the hippocampus of the newly-born SD rats was conducted for 10 days, then these cultured neurons that displayed seizure-like discharges in Mg2+-free medium were divided into normal extracellular fluid (medium) group (normal), Mg2+-free medium group, acupuncture serum group and non-acupuncture serum group (n=30). Blood examples were taken from pentylenetetrazol (i.p.i.) -induced acute convulsion adult SD rats underwent manual acupuncture stimulation of "Baihui" (GV 20) and "Dazhui" (GV 14, once daily for 7 days) to prepare acupuncture serum. Hippocampal neuronal cultures were prepared from hip-pocampal tissue isolated from 24 h-old SD rats. The isolated neurons were incubated normally in Dulbecco's Modified Eagle Me-dium (DMEM)/Nutrient Mixture F 12(1:1) for 10 days, followed by exposure to the extracellular fluid {composed of NaCl, KCl, CaCl2, MgCl2, HEPES[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], D-glucose and aminoethanoic acid (pH 7.3)} for 3 h and returned to normal culture for normal group, by exposure to Mg2+-free extracellular fluid (to induce seizure-like discharges) for 3 h and returned to normal culture for Mg2+-free medium group, by exposure to Mg2+-free extracellular fluid for 3 h and returned to a regular culture medium[DMEM/F 12(1:1)] plus acupuncture serum (9:1) for acupuncture serum group, and by exposure to Mg2+-free extracellular fluid for 3 h and returned to DMEM/F 12(1:1) plus non-acupuncture serum (9:1) for non-acupuncture serum group. The cell apoptosis was assessed at 2, 12 and 48 h after application of acupuncture serum or non-acupuncture serum by using TUNEL method and the expression levels of GRP 78, CHOP and Caspase-12 proteins of the cultured cells at the 3 time-points detected using Western blot.

RESULTS

The number of apoptotic hippocampal neurons was significantly higher in the Mg2+-free medium group than in the normal medium group at 2, 12 and 48 h (P<0.01), and considerably decreased in the acupuncture serum group (but not in the non-acupuncture serum group) relevant to the Mg2+-free medium group at the same time-points after application of acupuncture serum (P<0.05, P<0.01). The expression levels of GRP 78 protein at 2 h, CHOP and Caspase-12 proteins at 2, 12 and 48 h were significantly up-regulated in the Mg2+-free medium group relavant to the normal me-dium group (P<0.05, P<0.01). After application of acupuncture serum to the culture medium, the expression levels of GRP 78 protein at the three time-points were significantly increased in comparison with the Mg2+-free medium group (P<0.01,P<0.05), while those of CHOP at 12 and 48 h, and Caspase-12 at the three time-points were notably down-regulated (P<0.05,P<0.01). No significant differences were found between the non-acupuncture serum and Mg2+-free medium groups in the expression levels of GRP 78, CHOP and Caspase-12 proteins at the three time-points (P>0.05).

CONCLUSIONS

Acupuncture serum can significantly reduce apoptosis of the cultured hippocampal neurons, which may be related to its effects in increasing the expression of GRP 78 protein and down-regulating the expression of CHOP and Caspase-12 proteins, suggesting an important role of acupuncture serum in maintaining the stability of the neuronal endoplasmic reticulum.

Authors+Show Affiliations

Clinical College of Acu-moxibustion and Orthopedics-traumatology, Anhui University of Traditional Chinese Medicine, Hefei 230038, China.Clinical College of Acu-moxibustion and Orthopedics-traumatology, Anhui University of Traditional Chinese Medicine, Hefei 230038, China. saildoctor@126.com.College of Life Science, University of Science and Technology of China, Hefei 230012.Clinical College of Acu-moxibustion and Orthopedics-traumatology, Anhui University of Traditional Chinese Medicine, Hefei 230038, China.Clinical College of Acu-moxibustion and Orthopedics-traumatology, Anhui University of Traditional Chinese Medicine, Hefei 230038, China.Center for Scientific Research, Anhui University of Traditional Chinese Medicine, Hefei 230038, China.Clinical College of Acu-moxibustion and Orthopedics-traumatology, Anhui University of Traditional Chinese Medicine, Hefei 230038, China.Clinical College of Acu-moxibustion and Orthopedics-traumatology, Anhui University of Traditional Chinese Medicine, Hefei 230038, China.Clinical College of Acu-moxibustion and Orthopedics-traumatology, Anhui University of Traditional Chinese Medicine, Hefei 230038, China.

Pub Type(s)

Journal Article

Language

chi

PubMed ID

29071954

Citation

Zhang, Hui, et al. "[Protective Effect of Acupuncture Serum Derived From Acute Convulsion Rats On Cultured Hip-pocampal Neurons With Seizure-like Discharges By Regulating Expression of Endoplasmic Reticulum Stress-inducible Molecular Chaperones]." Zhen Ci Yan Jiu = Acupuncture Research, vol. 42, no. 2, 2017, pp. 95-101.
Zhang H, Yang F, Wu X, et al. [Protective Effect of Acupuncture Serum Derived from Acute Convulsion Rats on Cultured Hip-pocampal Neurons with Seizure-like Discharges by Regulating Expression of Endoplasmic Reticulum Stress-inducible Molecular Chaperones]. Zhen Ci Yan Jiu. 2017;42(2):95-101.
Zhang, H., Yang, F., Wu, X., Shen, D. K., Chen, H., Wu, S. B., Ang, W. P., Song, X. G., & Xie, M. Y. (2017). [Protective Effect of Acupuncture Serum Derived from Acute Convulsion Rats on Cultured Hip-pocampal Neurons with Seizure-like Discharges by Regulating Expression of Endoplasmic Reticulum Stress-inducible Molecular Chaperones]. Zhen Ci Yan Jiu = Acupuncture Research, 42(2), 95-101.
Zhang H, et al. [Protective Effect of Acupuncture Serum Derived From Acute Convulsion Rats On Cultured Hip-pocampal Neurons With Seizure-like Discharges By Regulating Expression of Endoplasmic Reticulum Stress-inducible Molecular Chaperones]. Zhen Ci Yan Jiu. 2017 Apr 25;42(2):95-101. PubMed PMID: 29071954.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Protective Effect of Acupuncture Serum Derived from Acute Convulsion Rats on Cultured Hip-pocampal Neurons with Seizure-like Discharges by Regulating Expression of Endoplasmic Reticulum Stress-inducible Molecular Chaperones]. AU - Zhang,Hui, AU - Yang,Fan, AU - Wu,Xu, AU - Shen,De-Kai, AU - Chen,Hao, AU - Wu,Sheng-Bing, AU - Ang,Wen-Ping, AU - Song,Xiao-Ge, AU - Xie,Ming-Yue, PY - 2017/10/27/entrez PY - 2017/10/27/pubmed PY - 2018/1/13/medline SP - 95 EP - 101 JF - Zhen ci yan jiu = Acupuncture research JO - Zhen Ci Yan Jiu VL - 42 IS - 2 N2 - OBJECTIVE: To observe the effect of acupuncture serum on the number of apoptosis of the cultured hippocampal neurons with seizure-like discharges and the expression levels of endoplasmic reticulum stress-inducible molecular chaperones glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP) and cysteine protease protein-12(Caspase-12), so as to reveal its protective mechanism on seizure-induced injury of hippocampal neurons. METHODS: A regular primary culture of neurons derived from the hippocampus of the newly-born SD rats was conducted for 10 days, then these cultured neurons that displayed seizure-like discharges in Mg2+-free medium were divided into normal extracellular fluid (medium) group (normal), Mg2+-free medium group, acupuncture serum group and non-acupuncture serum group (n=30). Blood examples were taken from pentylenetetrazol (i.p.i.) -induced acute convulsion adult SD rats underwent manual acupuncture stimulation of "Baihui" (GV 20) and "Dazhui" (GV 14, once daily for 7 days) to prepare acupuncture serum. Hippocampal neuronal cultures were prepared from hip-pocampal tissue isolated from 24 h-old SD rats. The isolated neurons were incubated normally in Dulbecco's Modified Eagle Me-dium (DMEM)/Nutrient Mixture F 12(1:1) for 10 days, followed by exposure to the extracellular fluid {composed of NaCl, KCl, CaCl2, MgCl2, HEPES[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], D-glucose and aminoethanoic acid (pH 7.3)} for 3 h and returned to normal culture for normal group, by exposure to Mg2+-free extracellular fluid (to induce seizure-like discharges) for 3 h and returned to normal culture for Mg2+-free medium group, by exposure to Mg2+-free extracellular fluid for 3 h and returned to a regular culture medium[DMEM/F 12(1:1)] plus acupuncture serum (9:1) for acupuncture serum group, and by exposure to Mg2+-free extracellular fluid for 3 h and returned to DMEM/F 12(1:1) plus non-acupuncture serum (9:1) for non-acupuncture serum group. The cell apoptosis was assessed at 2, 12 and 48 h after application of acupuncture serum or non-acupuncture serum by using TUNEL method and the expression levels of GRP 78, CHOP and Caspase-12 proteins of the cultured cells at the 3 time-points detected using Western blot. RESULTS: The number of apoptotic hippocampal neurons was significantly higher in the Mg2+-free medium group than in the normal medium group at 2, 12 and 48 h (P<0.01), and considerably decreased in the acupuncture serum group (but not in the non-acupuncture serum group) relevant to the Mg2+-free medium group at the same time-points after application of acupuncture serum (P<0.05, P<0.01). The expression levels of GRP 78 protein at 2 h, CHOP and Caspase-12 proteins at 2, 12 and 48 h were significantly up-regulated in the Mg2+-free medium group relavant to the normal me-dium group (P<0.05, P<0.01). After application of acupuncture serum to the culture medium, the expression levels of GRP 78 protein at the three time-points were significantly increased in comparison with the Mg2+-free medium group (P<0.01,P<0.05), while those of CHOP at 12 and 48 h, and Caspase-12 at the three time-points were notably down-regulated (P<0.05,P<0.01). No significant differences were found between the non-acupuncture serum and Mg2+-free medium groups in the expression levels of GRP 78, CHOP and Caspase-12 proteins at the three time-points (P>0.05). CONCLUSIONS: Acupuncture serum can significantly reduce apoptosis of the cultured hippocampal neurons, which may be related to its effects in increasing the expression of GRP 78 protein and down-regulating the expression of CHOP and Caspase-12 proteins, suggesting an important role of acupuncture serum in maintaining the stability of the neuronal endoplasmic reticulum. SN - 1000-0607 UR - https://www.unboundmedicine.com/medline/citation/29071954/[Protective_Effect_of_Acupuncture_Serum_Derived_from_Acute_Convulsion_Rats_on_Cultured_Hip_pocampal_Neurons_with_Seizure_like_Discharges_by_Regulating_Expression_of_Endoplasmic_Reticulum_Stress_inducible_Molecular_Chaperones]_ L2 - https://medlineplus.gov/seizures.html DB - PRIME DP - Unbound Medicine ER -